The gonadal steroid hormones, such as progesterone, are neuroprotective, but the receptors involved and the underlying mechanisms are still unclear. Historically, the field has focused primarily on the 'classical'intracellular receptors for these hormones, which operate as transcriptional regulators of gene expression. More recently, it has become apparent that these steroid hormones can elicit their effects through activation of membrane-associated receptors. The emerging neurobiology of these hormone receptors is in its infancy and as expected is surrounded by controversy. A serious limitation is the lack of appropriate and easily available molecular tools to selectively activate these receptors at the cell surface. Currently, the only available tools for distinguishing membrane-mediated effects of steroidal hormones from those mediated by their intracellular (classical) receptor(s) are the steroid-BSA conjugates that cannot enter into the cells because of their large size. However, due to shortcomings or caveats associated with these compounds (as outlined in Background &Significance section), there is a need for the development of additional/alternative molecular probes for studying steroid membrane receptors, which serves as the major driving force behind this proposal. Using progesterone as our starting point, our goal is to develop non-proteinaceous (abiotic), chemically stable and well-defined membrane-impermeable progesterone conjugates that can be used to distinguish membrane-mediated effects of the hormone from those mediated by the intracellular (classical) receptor(s). We will apply a well-established concept that ionic species cannot penetrate biological membranes in the absence of specific carrier macromolecules. Our hypothesis is that conjugation of a charged moiety to progesterone will preclude its entry into the cell and thus, result in activation of only membrane associated progesterone receptors. As such, this proposal focuses on the design, synthesis, stability studies (Specific Aims 1&2) of novel progesterone conjugates having a permanent positive charge in their molecular architecture. The ability of these conjugates to bind and activate a representative membrane-associated progesterone receptor (mPR) and therefore, initiate a non-classical signaling pathway will be investigated in Specific Aim 3. The pharmacological and neurobiological efficacy of the proposed membrane-impermeable conjugate will be further addressed in Specific Aim 4. PUBLIC HEALTH RELEVANCE: The recent controversy as to whether hormone therapy is beneficial or detrimental reflects, in part, our limited understanding of how such hormones as estrogen and progesterone influence the brain. With the emergence of newly recognized targets of these hormones (such as membrane progesterone receptors), there is a critical need to develop innovative tools to study and target these receptors. Successful completion of the proposed project will lead to the discovery of new compounds, whose long term benefit will enable the testing and development of safer and more effective ways of treating the menopause, and diseases whose risk increases following the menopause (including Alzheimer's disease).